This resubmitted Phase I SBIR application focuses on the development of a mucin-degrading colonic flora that competitively excludes Clostridium difficile, a gram-positive anaerobe that causes antibiotic-associated intestinal disease ranging from mild diarrhea to life-theatening pseudomembranous colitis. We are targeting C. difficile because this pathogen already is a major healthcare problem in the U.S., with over 300,000 cases per year and a relapse rate approaching 20%. In the event of massive antimicrobial prophylaxis, there will be a much larger population highly susceptible to infection with C. difficile. C. difficile also could be genetically manipulated to express other toxins such as the select agents, epsilon and botulinum toxins. Previous studies have shown that members of the Clostridium coccoides group degrade and utilize mucin, and competitively exclude C. difficile from the intestine. In Aim 1, we will identify and characterize potential members of a mucin-degrading flora from the coccoides group. We will identify the coccoides group members that most effectively metabolize mucins. The growth and maintenance procedures for the top mucin-degrading strains will be established. In Aim 2, we will determine the in vitro and in vivo performance characteristics of a mucin-degrading flora. The flora will be evaluated for protection and treatment efficacy in the hamster model of antibiotic-associated diarrhea and colitis. Members of an effective flora will be screened for the absence of transferable antibiotic resistance genes and toxin genes. In Phase 2, our studies will be extended to produce the mucin-degrading flora under GMP and initiate clinical studies with the flora under the direction of Dr. Kenneth Wilson at Duke University. Our goal is to determine if the mucin-degrading flora restores or preserves normal mucin metabolism, enhances resistance to colonization by C. difficile, and reduces the incidence of C. difficile- induced antibiotic-associated diarrhea. [unreadable] [unreadable] [unreadable]